Ningalin B analogs employable for reversing multidrug resistance

ABSTRACT

Anlogs of ningalin B lacking inherent cytotoxic activity may be employed to reverse multi-drug resistant (MDR) phenotype and to resensitize transformed cells, including a human colon cancer cell line (HCT116/VM46), with respect to a variety of cytotoxic agents, e.g., vinblastine and doxorubicin. In many instances, resensitization is achieved at lower doses than the prototypical agent verapamil. Total synthesis of ningalin B and its analogs was achieved using a concise, efficient approach based on a heterocyclic azadiene Diels-Alder strategy (1,2,4,5-tetrazine→1,2-diazine→pyrrole) ideally suited for construction of the densely functionalized pyrrole core found in the natural product is detailed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a US national phase application of international applicationSerial No. PCT/US01/06811, filed Mar. 1, 2001 and published in English,which claims priority from and is a continuation-in-part application ofU.S. provisional patent application Ser. No. 60/186,106, filed Mar. 1,2000.

This invention was made with United States Government support underContract No. CA 42056 from the National Institutes of Health. The UnitedStates Government has certain rights in the invention.

TECHNICAL FIELD

The invention relates to methods and reagents for reversing multidrugresistance (MDR) with respect to anticancer drugs. More particularly,invention relates to analogs of ningalin B and to their use as MDRreversal agents.

BACKGROUND

The recently identified ningalin class of marine natural productsincluding ningalin B (1) possess a common 3,4-diaryl substituted pyrrolenucleus bearing a 2-carboxylate. Ningalin B (1) is the second member ofthis newly described family of marine natural products which wereisolated by Fenical (1997) from an ascidian of the genus Didemnumcollected in western Australia near Ningaloo Reef. (Kang, H.; Fenical,W. J. Org. Chem. 1997, 62, 3254) Consequently, 1 and the relatedningalins are the newest members of a family of3,4-dihydroxyphenylalanine (DOPA)-derived o-catechol metabolites thatinclude the tunichromes. (Bruening, R. C.; et al. J. Am. Chem. Soc.1985, 107, 5289; Bruening, R. C.; et al. J. Nat. Prod. 1986, 49, 193;Bayer, E; et al. Angew. Chem. Int. Ed. Engl. 1992, 31, 52; Oltz, E. M.;et al. J. Am. Chem. Soc. 1988, 110, 6162; Ryan, D. E.; et al. J. Am.Chem. Soc. 1992, 114, 9659; Taylor, S. W.; et al. Arch. Biochem.Biophys. 1995, 324, 228)

The lamellarins are a related rapidly growing class of marine naturalproducts which were first isolated from the prosobranch molluscLamellaria sp. and important members of this class have been disclosedby Bowden, Faulkner, Fenical, Capon, and Scheuer. (Lamellarins A-D:Anderson, R. J.; et al. J. Am. Chem. Soc. 1985, 107, 5492. LamellarinsE-H: Lindquist, N.; et al. J. Org. Chem. 1988, 53, 4570. LamellarinsI-N: Carroll, A. R.; et al. Aust. J. Chem. 1993, 46, 489. Lamellarins O,P: Urban, S.; et al. Aust. J. Chem. 1994, 47, 1919. Lamellarins Q, R:Urban, S.; et al. Aust. J. Chem. 1995, 48, 1491. Lamellarins S: Urban,S.; et al. Aust. J. Chem. 1996, 49, 711. Lamellarins T-X: Reddy, R. M.;et al. Tetrahedron 1997, 53, 3457. Lamellarin Z: Davis, R. H.; et al. J.Nat. Prod. 1999, 62, 419. Lukianol A, B: Yoshida, W. Y.; et al. Helv.Chim. Acta 1992, 75, 1721.) Recent investigations of several lamellarinsdemonstrated their cytotoxic activity, revealed equally effectivecytotoxic activity against multidrug-resistant (MDR) cell lines, andrevealed MDR reversal even at noncytotoxic concentrations by inhibitionof P-glycoprotein (P-gp) mediated drug efflux. (Quesada, A. R.; et al.Br. J. Cancer 1996, 74, 677.) Thus, they constitute a new class ofantitumor agents which reverse MDR more effectively than verapamil andresensitize resistant malignant cells to front line therapeutics. Anumber of related structures have been defined that lack cytotoxicactivity but which effectively reverse MDR. (Ningalin A, lamellarin O,lukianol A, and permethyl storniamide A: Boger, D. L.; et al. J. Am.Chem. Soc. 1999, 121, 54.)

What is needed is a new class of MDR reversal agents having potentactivity for resensitizing resistant cancer cells with respect toeffective anticancer agents.

SUMMARY

A concise total synthesis of ningalin B (1) is described enlisting a1,2,4,5-tetrazine→1,2-diazine-pyrrole Diels-Alder strategy featuring theunusually effective [4+2] cycloaddition of the electron-deficient1,2,4,5-tetrazine 2 with an unsymmetrical, electron-rich alkyne.Ningalin B is a member of a class of marine natural productscharacterized by a highly functionalized tetra- or pentasubstitutedpyrrole which is ideally suited to construction using this strategy.While lacking inherent cytotoxic activity, the ningalin B syntheticprecursors 10, 11, 13, 14, and 15, but not ningalin B itself, are shownto potently reverse MDR, resensitizing a resistant human colon cancercell line (HCT116/VM46) to vinblastine and doxorubicin. These agents,including 14 bearing a novel ring system, constitute the members of anew class of effective MDR reversal agents.

More particularly, one aspect of the invention is directed to a compoundrepresented by the following structure:

wherein R is a radical selected from the group consisting of H and thefollowing structure:

Preferred embodiments of this aspect of the invention include either ofthe following structures:

Another aspect of the invention is directed to a compound represented bythe following structure:

wherein R is a radical selected from the group consisting of H, CO₂H,CO₂Me and CON(Me)₂. Preferred embodiments of this aspect of theinvention include compounds represented by the following structures:

Another aspect of the invention is directed to an analog of ningalin Brepresented by the following structure:

Another aspect of the invention is directed to a synthetic processcomprising the step of cyclizing a precursor compound with an excess ofEaton's acid at room temperature under reaction conditions for producingan analog of ningalin B, the precursor compound, the analog of ningalinB, and the cyclization reaction being represented as follows:

Another aspect of the invention is directed to a process for reversingmultidrug resistance in a cancer cell. The process comprises the step ofcontacting the cancer cell with a concentration sufficient for reversingsaid multidrug resistance of a compound selected from a group consistingof any or all of the following structures:

BRIEF DESCRIPTION OF FIGURES

FIG. 1 illustrates the structure of the natural product ningalin B.

FIG. 2 illustrates a retrosynthetic scheme for synthesizing ningalin Band its analogs.

FIG. 3 illustrates the first portion of the synthetic scheme used tosynthesize ningalin B.

FIG. 4 is a continuation of FIG. 3 and illustrates the completion of thesynthesis of ningalin B.

FIG. 5 illustrates the product obtained from an attempteddecarboxylation of structure 12 using Eaton's acid.

FIG. 6 illustrates a table with a comparison of the cytotoxic activityof ningalin B and some analogs with some commonly used compounds againstthree different cell lines.

FIG. 7 illustrates a table with a comparison of the ability of ningalinB and its analogs to reverse multidrug resistance in the HCT116/VM46cell line.

FIG. 8 illustrates the inhibition of dye efflux (rhodamine 123) from theHCT116/VM46 cell line. Accumulation of rhodamine 123 in the HCT116/VM46cell line after 30 min incubation in 40 mM rhodamine in phosphate buffersolution (Quesada, A. R.; et al., Br. J. Cancer 1996, 74, 677).

FIG. 9 illustrates the structures of the analogs and ningalin B.

FIG. 10 illustrates data on the cytotoxicity of compounds 14 and 15against four cancer cell lines, according to the method of FIG. 6.

FIG. 11 illustreates further data on the reversal of multidrugresistance (MDR) by compounds 14 and 15 with respect to vinblastine anddoxorubicin against the cell line HCT116/VM46.

DETAILED DESCRIPTION

The total synthesis of ningalin B (1) and a number of structurallyrelated synthetic analogs is described herein. Also described herein isa biological evaluation of the natural product and its syntheticanalogs. The synthetic approach, complementary to the efforts describedto date, (Lukianol A and lamellarin O dimethyl ether: Fürster, A.; etal. J. Org. Chem. 1995, 60, 6637. Lamellarin O and Q, lukianol A:Banwell, M. G.; et al. Chem. Commun. 1997, 207. Lamellarin K: Banwell,M.; et al. Chem. Commun. 1997, 2259. Lamellarin D and H: Ishibashi, F.;et al. Tetrahedron 1997, 53, 5951. Lamellarin G trimethyl ether: Heim,A.; et al. Angew. Chem. Int. Ed. Engl. 1997, 36, 155. Storniade Anonamethyl ether: Ebel, H.; et al. Tetrahedron Lett. 1998, 39, 9165.Polycitrin A: Terpin, A.; et al. Tetrahedron 1995, 51, 9941.) employs aheteroaromatic azadiene Diels-Alder reaction (Boger, D. L. Chemtracts:Org. Chem. 1996, 9, 149. Boger, D. L. Bull. Clim. Soc., Belg. 1990, 99,599. Boger, D. L.; et al. In Progress in Heterocyclic Chem. 1989;Suschitzky, H.; Scriven, E. F. V., Eds.; Pergamon: Oxford, Vol. 1; 1989,30. Boger, D. L.; et al. Hetero Diels-Alder Methodology in OrganicSynthesis; Academic: San Diego, 1987. Boger, D. L.; et al. Chem. Rev.1986, 86, 781. Boger, D. L. Tetrahedron 1983, 39, 2869.) to assemble thesubstituents onto a six-membered 1,2-diazine core which is followed by areductive ring contraction reaction (Boger, D. L.; et al. J. Org. Chem.1984, 49, 4405. Boger, D. L.; et al. J. Org. Chem. 1988, 53, 1405.Boger, D. L.; et al. J. Am. Chem. Soc. 1993, 115, 11418. Boger, D. L.;et al. J. Org. Chem. 1985, 50, 5377. Boger, D. L.; Org. Syn. 1991, 70,79.) to provide the corresponding pyrrole (FIG. 2).

Total Synthesis of Ningalin B. The requisite diphenylacetylene 5 wasprepared by a palladium(0)-catalyzed cross-coupling of the terminalacetylene 3 (Upasami, R. B.; et al. J. Med. Chem. 1997, 40, 73.) and 4(0.05 equiv Pd(0), 0.3 equiv CuI, Et₃N, 87%) in which slow addition ofthe acetylene was necessary to suppress competitive formation of thecoupled diacetylene (FIG. 3). Conversion to the methoxymethyl ether 6was accomplished by Baeyer-Villiger oxidation of aldehyde 5 (1.2 equivm-CPBA), formate hydrolysis (KOH), and subsequent protection of thephenol (3.0 equiv MOMCl, 4.0 equiv i-Pr₂NEt, 67% overall). The first ofthe two key conversions, the Diels-Alder reaction of the electron-richacetylene 6 with the electron-deficient 1,2,4,5-tetrazine 2, (Boger, D.L.; et al. J. Org. Chem. 1985, 50, 5377. Boger, D. L.; et al. Org.Synth. 1991, 70, 79.) proceeded to give the desired 1,2-diazine 7 inexcellent yield (mesitylene, 140° C., 92%). The relative facility ofthis inverse electron demand [4+2] cycloaddition may be attributed tothe electron-donating properties of the dienophile aryl alkoxy groups.Thus, the oxygenation pattern found in the diaryl acetylene 6 increasesthe nucleophilic character and improves what is a typically poorreactivity of an alkyne towards 2. (Sauer, J.; et al. Chem. Ber. 1965,98, 1435) Subsequent reductive ring contraction (Zn, HOAc, 62%) of 7afforded the core pyrrole structure found in the natural product.N-Alkylation with the phenethyl bromide 9 (Lan, A. J. Y.; et al. J. Am.Chem. Soc. 1987, 109, 2738) (5.0 equiv, K₂CO₃, 94%) and subsequent MOMdeprotection with concomitant lactonization (HCl-EtOAc, 95%) providedmono-lactone 11. (FIG. 4) Selective hydrolysis of the methyl ester (LiI,80%) and decarboxylation (Cu₂O, quinoline, 220° C., 5 min, 70%) affordedhexamethyl ningalin B (13). Decarboxylation with alternative coppersources or those conducted at lower temperatures or with longer reactiontimes resulted in lower yields (0-44%). Exhaustive demethylation withBBr₃ completed the total synthesis of ningalin B and provided materialidentical in all respects (¹H NMR, ¹³C NMR, IR, MS) with authenticmaterial. (Kang, H.; Fenical, W. J. Org. Chem. 1997, 62, 3254.)

Initial attempts to promote decarboxylation under acidic conditionsresulted in either no reaction (neat TFA, 60° C., 12 h) orFriedel-Crafts acylation (neat Eaton's acid, 25° C., 18 h) to provide 14(FIG. 5). Although not the object of the present efforts, the fusedtricyclic ring system consisting of a 7-membered ketone flanked by anaryl group and a pyrrole has been formed by Friedel-Crafts acylation inthe synthesis of cephalotaxus alkaloids. (Girard, Y.; et al. J. Org.Chem. 1983, 48, 3220. Weinstein, B.; et al. J. Org. Chem. 1976, 41,875.) Based on the precedented ease of formation of the 7-membered ringand ¹H and HMBC NMR spectroscopy, formation of the alternative5-membered ring was excluded. Importantly, 14 proved to be the mostpotent MDR reversal agent identified in this series, causinghypersensitivity towards vinblastine in the HCT/VM46 MDR-cell line.

Cytotoxic Activity and Reversal of Multidrug Resistance. A number ofcompounds in the structurally related lamellarin class of naturalproducts possess cytotoxic activity. (Quesada, A. R.; et al. Br. J.Cancer 1996, 74, 677.) With exception of ningalin A, (Ningalin A,lamellarin O, lukianol A, and permethyl storniamide A: Boger, D. L.; etal. J. Am. Chem. Soc. 1999, 121, 54.) the biological evaluation of theningalin family has not been explored. Consequently, ningalin B and anumber of structurally related synthetic intermediates were tested in aL1210 cytotoxic assay, and the results are summarized in FIGS. 6 and 10.Ningalin B was found to be only moderately active against both the L1210and HCT116 cell lines, and a number of synthetic intermediates displayeda similar level of activity due to their comparable structures. Notably,the O-methyl derivative of ningalin B is 5-fold less active againstL1210 and 2.5-fold less active against HCT116 than ningalin B, inagreement with previous studies where an increase in the extent ofO-methylation results in a decrease in cytotoxic activity. (Ningalin A,lamellarin O, lukianol A, and permethyl storniamide A: Boger, D. L.; etal. J. Am. Chem. Soc. 1999, 121, 54.)

More importantly, a select set of the naturally occurring lamellarinshave been shown to exhibit equally potent cytotoxic activity againstmultidrug resistant (MDR) cell lines due to overexpression ofP-glycoprotein and to reverse MDR at noncytotoxic concentrations,resensitizing the resistant cell lines to conventional therapeuticagents. (Quesada, A. R.; et al. Br. J. Cancer 1996, 74, 677.) P-gp is a170 kDa plasma membrane glycoprotein encoded in humans by the MDR1 genewhich exports drugs out of mammalian cells, lowering their intracellularconcentration. (Patel, N. H.; et al. Invest. New Drugs 1994, 12, 1.Gottesman, M. M.; et al. Annu. Rev. Biochem. 1993, 62, 385.) Therefore,7-14 were also tested against a wild-type human colon cancer cell line(HCT116) and two resistant HCT116 cell lines. The first resistant cellline (HCT116/VM46) embodies the MDR phenotype and overexpressesP-glycoprotein while the second cell line (HCT116/VP35) derives itsresistance through underexpression of topoisomerase II. The examinationof the latter cell line along with the wild-type HCT116 and theircomparison with HCT116VM46 allows an accurate assessment of the MDRsensitivity as well as an assessment of one potential therapeutictarget. All of the agents examined showed little or no intrinsiccytotoxic activity against either HCT116 or the resistant cell lines.

Fundamentally more important, many of the agents were found capable ofreversing MDR at noncytotoxic concentrations, resensitizing HCT116/VM46to vinblastine and doxorubicin (FIGS. 7 and 11). As illustrated in FIGS.7 and 11, solutions of physiological buffer suitable for injection orinfusion were prepared and admixed with ningalin B and its analogs fortesting as MDR reversal agents. Of the compounds examined, 10, 11, 13,and 14 were able to resensitize HCT116/VM46 to vinblastine anddoxorubicin at 1 μM and to do so more effectively than verapamil. Whilelacking inherent cytotoxicity, 11 and 13 showed complete MDR reversal atthis concentration and 14 caused hypersensitivity of HCT116/VM46 tovinblastine, exhibiting an IC₅₀ value 3× lower than wild type treatmentwith vinblastine alone. At the higher concentrations required forcomplete reversal by verapamil (7.5 μM), 10 showed complete MDR reversaland the HCT116/VM46 cell line became hypersensitive to vinblastine inthe presence of 11 and 13. The HCT116/VP35 resistant cell line showed noresensitization towards vinblastine or doxorubicin in the presence ofthe examined agents, indicating that the MDR reversal activity is due tointeraction with P-gp. Consistent with its action on Pgp-170, 14inhibited dye efflux (Quesada, A. R.; et al. Br. J. Cancer 1996, 74,677.) (rhodamine 123) from HT116/VM46, returning the dye retention tolevels equivalent to that of wild type HCT116 (FIG. 8).

EXAMPLES

2-[(3,4-Dimethoxyphenyl)ethynyl]-4,5-dimethoxybenzaldehyde (5). Astirred solution of 4 (2.7 g, 11 mmol, 1.0 equiv), PdCl₂(PPh₃)₂ (0.39 g,0.55 mmol, 0.05 equiv) and CuI (0.63 g, 3.31 mmol, 0.3 equiv) in 5:1DMF-Et₃N (106 mL) under Ar at 75° C. was treated with 3¹² (2.23 g, 13.8mmol, 1.25 equiv) in 5:1 DMF-Et₃N (42 mL) over a period of 2.5 h. Thereaction mixture was allowed to stir for an additional 1.5 h before itwas cooled to 25° C. and concentrated under reduced pressure.Chromatography (SiO₂, 4.5 (20 cm, CH₂Cl₂) afforded 5 (1.50 g, 87% yield)as a yellow solid: mp 148-149° C. (EtOAc-hexanes); FABHRMS (NBA/NaI) m/z327.1228 (M+H⁺, C₁₉H₁₈O₅ requires 327.1232).

2-[(3,4-Dimethoxyphenyl)ethynyl]-4,5-dimethoxy-1-(methoxymethoxy)-benzene(6). A stirred solution of 5 (3.13 g, 9.60 mmol, 1.0 equiv) in CH₂Cl₂(380 mL under Ar at 25° C. was treated with Na₂HPO₄ (3.27 g, 23.03 mmol,2.4 equiv) and m-CPBA (3.98 g, 11.52 mmol, 1.2 equiv). After 18 h, themixture was diluted with saturated aqueous NaHCO₃, extracted with EtOAc,washed with saturated aqueous NaHCO₃ and saturated aqueous NaCl, dried(Na₂SO₄), and concentrated under reduced pressure. The formate wasredissolved in MeOH (120 mL), treated with 10% aqueous KOH (7.8 mL, 15.6mmol, 1.6 equiv), and the mixture was stirred at 25° C. for 1.5 h. Thereaction was quenched with the addition of 10% aqueous HCl, extractedwith CH₂Cl₂, washed with H₂O, dried (Na₂SO₄), and the solvent wasremoved under reduced pressure. An analytically pure sample of thephenol could be prepared by chromatography (SiO₂, 5% EtOAc/CH₂Cl₂): mp164-165° C. (EtOAc-hexanes); MALDIHRMS (DHB) m/z 337.1058 (M+Na⁺,C₁₈H₁₈O₅ requires 337.1046). A solution of the crude phenol in CH₂Cl₂(100 mL) under Ar at 0° C. was treated with ^(i)Pr₂NEt (6.70 mL, 38.4mmol, 4.0 equiv) and chloromethyl methyl ether (2.19 mL, 28.8 mmol, 3.0equiv). The mixture was warmed to 25° C. and allowed to stir for 18 h.Following dilution with H₂O, the mixture was extracted with CH₂Cl₂,washed with saturated aqueous NaHCO₃, saturated aqueous NaCl, dried(Na₂SO₄), and concentrated under reduced pressure. Chromatography (SiO₂,4.5 (15 cm, 5% EtOAc/CH₂Cl₂) afforded 6 (2.32 g, 67% yield) as an orangesolid: mp 84-86° C. (EtOAc-hexanes); MALDIHRMS (DHB) m/z 358.1411 (M⁺,C₂₀H₂₂O₆ requires 358.1416).

Dimethyl4-(4,5-Dimethoxy-2-(methoxymethoxy)phenyl)-5-(3,4-dimethoxyphenyl)-1,2-diazine-3,6-dicarboxylate(7). A solution of 6 (1.10 g, 3.07 mmol, 1.0 equiv) and3,6-dicarbomethoxy-1,2,4,5-tetrazine (2,¹³ 0.91 g, 4.60 mmol, 1.5 equiv)in mesitylene (15.4 mL) was warmed at 140° C. under Ar for 24 h.Additional 2 (0.91 g, 4.60 mmol, 1.5 equiv) was added, and the mixturewas maintained at 140° C. for an additional 24 h before the reactionmixture was cooled to 25° C. and the solvent was evaporated.Chromatography (SiO₂, 4.5 (20 cm, 30% EtOAc/CH₂Cl₂) provided 7 (1.49 g,92% yield) as an orange oil. An analytically pure sample was prepared byrecrystallization from EtOAc-hexanes: mp 131-133° C.; FABHRMS (NBA/NaI)m/z 551.1663 (M+Na⁺, C₂₆H₂₈N₂O₁₀ requires 551.1642).

Dimethyl3-(4,5-Dimethoxy-2-(methoxymethoxy)phenyl)-4-(3,4-dimethoxyphenyl)pyrrole-2,5-dicarboxylate(8). A solution of 7 (1.01 g, 1.91 mmol, 1.0 equiv) in HOAc (25 mL)under Ar at 25° C. was treated with activated Zn dust (1.25 g, 19.1mmol, 10 equiv), stirred for 4 h, and then treated with additional Zndust (1.25 g, 10 equiv). After 14.5 h, the slurry was diluted with 10%MeOH/CHCl₃ (25 mL) and stirred 3 h at 25° C. The mixture was filteredthrough Celite, rinsed with 10% MeOH/CHCl₃, and the filtrate was washedwith saturated aqueous NaHCO₃, dried (Na₂SO₄), and concentrated invacuo. Chromatography (SiO₂, 4.5×15 cm, 25% EtOAc/CH₂Cl₂) afforded 8(0.61 g, 62% yield) as an orange oil. An analytically pure sample couldbe prepared by recrystallization from EtOAc-hexanes: mp 162-163° C.;MALDIHRMS (DHB) m/z 515.1800 (M⁺, C₂₆H₂₉NO₁₀ requires 515.1791).

Dimethyl3-(4,5-Dimethoxy-2-(methoxymethoxy)phenyl)-4-(3,4-dimethoxyphenyl)-1-[2-(3,4-dimethoxyphenyl)ethyl]pyrrole-2,5-dicarboxylate(10). A stirred mixture of 8 (297 mg, 0.58 mmol, 1.0 equiv),3,4-dimethoxyphenethyl bromide (9,¹⁵ 707 mg, 2.88 mmol, 5.0 equiv), andK₂CO₃ (398 mg, 2.88 mmol, 5 equiv) in DMF (5.8 mL) under Ar was warmedto 70° C. After 2.5 h, the mixture was cooled to 25° C. and solvent wasremoved in vacuo. Chromatography (SiO₂, 3.5×15 cm, 20% EtOAc/CH₂Cl₂)provided 10 (372 mg, 94% yield) as a yellow oil: FABHRMS (NBA/NaI) m/z702.2553 (M+Na⁺, C₃₆H₄₁NO₁₂ requires 702.2526).

Methyl7,8-Dimethoxy-3-(2-(3,4-dimethoxyphenyl)ethyl)-1-(3,4-dimethoxyphenyl)-[1]-benzopyrano[3,4-b]pyrrol-4(3B)-one-2-carboxylate(11). A sample of 10 (272 mg, 400 μmol, 1.0 equiv) was treated with 3 MHCl-EtOAc (16 mL) and stirred under Ar at 25° C. for 2 h. Chromatographyof the concentrated mixture (SiO₂, 4.5×5 cm, 15% EtOAc/CH₂Cl₂) affordedpure 11 (229 mg, 95%) as a light yellow solid: mp 192-193° C.; FABHRMS(NBA/NaI) m/z 626.2017 (M+Na⁺, C₃₃H₃₃NO₁₀ requires 626.2002).

7,8-Dimethoxy-3-(2-(3,4-dimethoxyphenyl)ethyl)-1-(3,4-dimethoxyphenyl)-[1]-benzopyrano[3,4-b]pyrrol-4(3B)-one-2-carboxylicAcid (12). A stirred mixture of 11 (120 mg, 0.20 mmol, 1.0 equiv) andLiI (80 mg, 0.60 mmol, 3.0 equiv) in DMF (13 mL) under Ar was warmed atreflux. After 24 and 48 h, the reaction was treated with additional LiI(80 mg, 2×3 equiv). The mixture was warmed for a total of 3.5 d beforethe reaction was diluted with H₂O, acidified with 10% aqueous HCl,extracted with CH₂Cl₂, and dried (Na₂SO₄). Chromatography (SiO₂, 2.0×15cm, 5% MeOH/CHCl₃) afforded 12 (94 mg, 80% yield) as a yellow solid: mp219-220° C.; MALDIHRMS (DHB) mn/z 589.1940 (M⁺, C₃₂H₃₁NO₁₀ requires589.1948).

Hexamethyl Ningalin B (13). A solution of 12 (9.3 mg, 16 μmol, 1.0equiv) and cuprous oxide¹⁸ (2.3 mg, 16 μmol, 1.0 equiv) in degassedquinoline (450 μL) was warmed at 220° C. under Ar for 5 min. The mixturewas cooled to 25° C., and the solvent was removed by a stream of N₂.Chromatography (SiO₂, 0.5×10 cm, 10% EtOAc/CH₂Cl₂) provided 13 (6.0 mg,70% yield) as a white solid: mp 186-187° C.; MALDIHRMS (DHB) mn/z546.2111 (M+H⁺, C₃₁H₃₁NO₈ requires 546.2128).

Ningalin B (1). A solution of 13 (5.9 mg, 11 μmol, 1.0 equiv) in CH₂Cl₂(1.1 mL) under Ar at −78° C. was treated with BBr₃ (1 M in hexanes, 160μL, 160 μmol, 15 equiv), and the mixture was allowed to warm to 25° C.over 24 h. Following dilution with MeOH (0.50 mL), the solvent wasremoved by a stream of N₂ to afford synthetic 1 (5.2 mg, 98%) identicalin all respects (¹H NMR, ¹³C NMR, IR, MS) when compared to spectra ofnaturally derived ningalin B: MALDIHRMS (DHB) m/z 484.1009 (M+Na⁺,C₂₅H₁₉NO₈ requires 484.1008).

9,10-Dihydro-12,13-dimethoxy-1-(3′,4′-dimethoxyphenyl)-3,4-dimethoxy-[4,3-d]-[1]-benzopyrano-15H-benzazepino[3,2-a]-[3]-pyrrol-7,15(18H)-dione(14). A sample of 12 (3.3 mg, 5.6 μmol, .1.0 equiv) was treated withEaton's Acid¹⁹ (200 μL, 7.5% P₂O₅—MeSO₃H) and stirred under Ar at 25° C.After 18 h, the reaction was diluted with H₂O, extracted with CH₂Cl₂,washed with saturated aqueous NaHCO₃ and saturated aqueous NaCl, dried(Na₂SO₄), and concentrated under reduced pressure. Chromatography (SiO₂,1.5×5 cm, 10% EtOAc/CH₂Cl₂) afforded 14 (2.1 mg, 66% yield) as a yellowsolid: mp 225-226° C.; MALDIHRMS (DHB) m/z 572.1940 (M+H⁺, C₃₂H₂₉NO₉requires 572.1921).

N,N-Dimethyl7,8-Dimethoxy-3-(2-(3,4-dimethoxyphenyl)ethyl)-1-(3,4-dimethoxyphenyl)-[1]-benzopyrano[3,4-b]pyrrol-4(3H)-one-2-carboxamide(15). A solution of 12 (58.1 mg, 0.098 mmol, 1.0 equiv), EDCI (37.5 mg,0.196 mmol, 2.0 equiv), and HOBt (26.5 mg, 0.196 mmol, 2.0 equiv) inCH₂Cl₂ (4 mL) under Ar at 25° C. was treated with (CH₃)₂NH (2M in THF,735 μL, 1.47 mmol, 15 equiv). After 16 h, the solvent was removed andchromatography (SiO₂, 1.5×12 cm, 1% MeOH/CHCl₃) afforded pure 15 (58.5mg, 97% yield) as a white glass: MALDIHRMS (DHB) m/z 617.2500 (M+H⁺,C₃₄H₃₆N₂O₉ requires 617.2494).

1. A compound represented by the following structure:

wherein R is a radical selected from the group consisting of H and thefollowing structure:


2. A compound according to claim 1 represented by the followingstructure:


3. A compound according to claim 1 represented by the followingstructure:


4. A compound represented by the following structure:

wherein R is a radical selected from the group consisting of H, CO₂H,CO₂Me and CON(Me)₂.
 5. A compound according to claim 4 represented bythe following structure:


6. A compound according to claim 4 represented by the followingstructure:


7. A compound according to claim 4 represented by the followingstructure:


8. A compound according to claim 4 represented by the followingstructure:


9. An analog of ningalin B represented by the following structure:


10. A synthetic process comprising the following step: cyclizing aprecursor compound with an excess of Eaton's acid at room temperatureunder reaction conditions for producing an analog of ningalin B, theprecursor compound, the analog of ningalin B, and the cyclizationreaction being represented as follows:


11. A process for P-gp mediated reversing multidrug resistance in acancer cell comprising the step of contacting said cancer cell with aconcentration sufficient for reversing said P-gp mediated multidrugresistance of a compound selected from a group consisting of compoundsrepresented by the following structures:


12. A process according to claim 11 wherein the compound is representedby the following structure:


13. A process according to claim 11 wherein the compound is representedby the following structure:


14. A process according to claim 11 wherein the compound is representedby the following structure:


15. A process according to claim 11 wherein the compound is representedby the following structure:


16. A process according to claim 11 wherein the compound is representedby the following structure:


17. A process according to claim 11 wherein the compound is representedby the following structure:


18. A process according to claim 11 wherein the compound is representedby the following structure:


19. A process according to claim 11 wherein the compound is representedby the following structure:


20. A solution comprising a physiological buffer suitable for injectionor infusion admixed a P-gp mediated multidrug resistance reversal agenthaving a concentration sufficient to resensitize a cancer cell having aresistance to an anticancer drug, the multidrug resistance reversalagent being selected from any of the compounds described in claim 9.